Human subjects exposed to a condition or disease offer a source of antibodies with therapeutic potential and general methods for obtaining such antibodies are known in the art. However, methods for specifically obtaining antibodies with therapeutic potential are generally limited by the low frequency, slow proliferation rate, and low antibody secretion levels of B cells that express such antibodies. For example, memory B cells with defined specificity typically account for only one cell per million peripheral blood mononuclear cells or approximately one milliliter of blood (Lanzavecchia et al., Curr. Opin. Immunol., 21:298-304 (2009): Yoshida et al., Immunol. Rev., 237:117-139 (2010)). The frequency of antibodies with therapeutic potential is likely to be even lower in cancer patients, necessitating the development of novel approaches that enable isolation of such cells with high sensitivity and efficiency.
Conventional methods generally rely on conversion of memory B cells into antibody secreting cells by in vitro culture and/or use of immunized animal models (e.g., mice) (Crotty et al., J. Immunol., 171:4969-4973 (2003): Fecteau et al., Immunology, 128:e353-e365 (2009): Buisman et al., Vaccine, 28:179-186 (2009): Corti et al., PLoS One, 5:e8805 (2010)). For example, following in vitro culture for up to one week, antibodies can be measured in culture supernatants and frequencies of antibody secreting cells assessing using enzyme-linked immunosorbent spot (ELISPOT) assay. Limitations of such methods are reported (Henn et al., J. Immunol., 183:31777-3187 (2009): Cao et al., J. Immunol., Methods, 358:56-65 (2010)). For instances, in vitro culture of memory B cells alters the memory B cell phenotype to resemble plasma cells with distinct functional properties (Jiang et al., Eur. J. Immunol., 37:2205-2213 (2007): Huggins et al., Blood, 109:1611-1619 (2007): Jourdan et al., Blood, 114:5173-5181 (2009)). Limitations for fluorescent antigen-based methods are also reported (Hofer et al., Immunol. Rev., 211:295-302 (2006): Odendahl et al., Blood, 105:1614-1621 (2005); Kunkel et al., Nat. Rev. Immunol., 3:822-829 (2003): Scheid et al., Nature, 458:636-640 (2009): Wu et al., Science, 329:856-861 (2010)).
Improved methods for specifically obtaining or targeting antibodies with therapeutic potential are required.
MICA is a ligand for NKG2D, a C-type lectin-like, type II transmembrane receptor expressed on most human NK cells, γδ T cells, and CD8+ T cells. Upon ligation, NKG2D signals through the adaptor protein DAP10 to evoke perforin dependent cytolysis and to provide co-stimulation. In humans, the NKG2D ligands include MHC class I chain-related protein A (MICA), the closely related MICB, UL-16 binding proteins (ULBP) 1-4, and RAE-1G. While NKG2D ligands are not usually found on healthy tissues, various forms of cellular stress, including DNA damage, may upregulate ligand expression, resulting in their frequent detection in multiple solid and hematologic malignancies, including melanoma. NKG2D activation through ligand positive transformed cells contributes to extrinsic tumor suppression, since NKG2D deficient and wild type mice treated with anti-NKG2D blocking antibodies manifest enhanced tumor susceptibility. Immune escape may be achieved in patients, however, by the shedding of NKG2D ligands from tumor cells, which triggers internalization of surface NKG2D and impaired function of cytotoxic lymphocytes. Soluble NKG2D ligands may also stimulate the expansion of regulatory NKG2D+CD4+Foxp3− T cells that may antagonize anti-tumor cytotoxicity through Fas ligand, IL-10, and TGF-β. MICA is a NKG2D ligand shed from tumor cells, i.e., released from the cell surface into the surrounding medium, and sera from cancer patients typically contain elevated levels of the soluble form (sMICA). MICA shedding is accomplished in part through interactions with the protein disulfide isomerase ERp5, which forms a disulfide bond with a critical cysteine that results in unfolding of the α3 domain, rendering it susceptible to proteolysis by ADAM-10/17 and MMP14.
Angiogenesis is the process of forming new capillaries from preexisting blood vessels and has been implicated as a critical part of tumor growth and dissemination. Tumors stimulate angiogenesis to meet increasing oxygen and nutrient requirements that exceed those that can be met by diffusion alone. Consequently, tumors recruit, remodel and expand existing vascular to meet their metabolic demand. The dependence of growing tumors on new blood vessel formation has made angiogenesis an appealing target for anti-cancer therapies. Many cytokines have been are believed to play a role in the regulation of angiogenesis, including vascular endothelial growth factor (VEGF) family members and the angiopoietins. The angiopioetins were discovered as ligands for the Ties, a family of tyrosine kinases that is selectively expressed in the vascular endothelium. There are four know angiopoietins: angiopoietin-1 (“Ang-1”) through angiopoietin-4 (“Ang-4”). Studies have suggested that angiopoietins (e.g., Ang-1 and Ang-2) may be involved and tumor angiogenesis. With this information, angiopoietins have been identified as potential targets of immune-based cancer therapy.
There is a need to identify new agents that specifically recognize and bind targets of immune-based cancer therapy, such as MICA and angiopoietins. Such agents would be useful for diagnostic screening and therapeutic intervention in disease states that are associated with tumor development.